The use of semi- and fully-automated analyzer systems for the quantification, identification, and characterization of the cells in whole blood aids in the efficiency and economy of performing all types of hematology analyses known in the art. Automated systems are used in the analysis of a variety of both normal and abnormal counterparts of all blood cell types, for example, red blood cells (erythrocytes), reticulocytes (immature erythrocytes), white blood cells (leukocytes), platelets, and in the determination of many parameters related thereto. For example, mean cell volume and hemoglobin concentration and content are characteristics of red blood cells which are routinely measured.
Problems in the art arise due to lack of accuracy, precision, and reproducibility of the final results, which may stem from cell contamination due to inefficient and/or suboptimal methods and reagents, as well as from accumulated build-up of reagents in the sample chambers and system hardware, especially when hundreds of samples are processed and analyzed in rapid fashion.
Although automated hematology processes and automated flow systems therefor have been developed to ease the burden of all types of blood cell analysis, the processes and systems must be continously cleaned and kept in optimal working order to insure reliable, accurate, and efficient operativity and results. Specific examples of the various types of hematology analyses, methods, and improvements thereof, that are performed using automated systems include differential white blood cell counting, such as described in U.S. Pat. No. 3,741,875 to Ansley et al.; U.S. Pat. No. 4,099,917 to Kim; and U.S. Pat. Nos. 4,801,549 and 4,978,624 to Cremins et al.; blood cell and reticulocyte analyses, including the analysis of a variety of characteristics of these cell types, such as described, for example, in U.S. Pat. No. 5,350,695 to G. Colella et al., U.S. Patent Nos. 5,360,739 and 5,411,891 to S. Fan et al., U.S. Pat. No. 4,735,504 to Tycko, and in C. Brugnara et al., 1994, Am. J. Clin. Path., 102(5):623-632. Many of the automated hematology systems rely on electrooptical measurements and absorbance/light scatter or fluorescence/light scatter flow cytometry techniques to obtain and present the final results as cytograms or useful output.
In general, for the performance of each type of hematology analysis performed using an automated system, the system is designed to have several different channels which are responsible for carrying out distinct functions during the analysis and/or displaying information about individual cell types or the particular parameters being measured. As a consequence of the various operational channels and the numbers of samples to be routinely processed, the automated systems, including all sample chambers, and the system hardware, including channels, pumps, tubing, valves, containers, joints, and the like, must be routinely rinsed with one or more reagent solutions to avoid build-up and contamination by the reaction mixture, which comprises the blood sample and other reagent components, after repeated aspirations of the samples and the sample reagent solutions undergoing rapid analysis. A particular example is demonstrated by current automated hematology analyzer systems, such as those commercially available under the trade designation TECHNICON H.circle-solid..TM., e.g., H.circle-solid.1.TM., H.circle-solid.2.TM., and H.circle-solid.3.TM., and the like, and sold by the assignee of the present invention, in which more than one type of rinse reagent is used, depending on the type of analysis being performed. As more particularly exemplified, the aforementioned hematology systems generally require one type of rinse reagent formulation for the hemoglobin (i.e., Hb) and red blood cell/platelet (i.e., RBC/PLT) channels, and a different type of rinse reagent for the peroxidase and basophil channels used in performing hematology analyses.
As indicated, problems and interference result from the carryover of one reaction mixture into another reaction mixture in the chambers of automated analyzers, particularly when the chambers are used again and again for multiple sample analyses. By reaction mixture is meant a whole blood sample mixed with a reagent composition comprising appropriate concentrations or amounts of reagent components.
Rinse solution carryover, when it contributes reactive chemical components, such as lytic surfactant, to a reaction mixture undergoing analysis in a method (i.e., when it "participates" in any way in a reaction mixture) may adversely affect the analytical results and lead to erroneous, inaccurate, and imprecise determinations and cytogram readings. That rinse carryover varies from system to system also presents problems and adversely affects the results of and information obtained from an analytical method. For example, too little rinse carryover volume in a method can result in excess noise at the origin of cytograms. Alternatively, excess rinse carryover volume in a method can cause the white blood cells to be attacked by the presence of active lytic surfactant in the carryover volume. Such adverse effects compromise the accuracy and reliability of hematological results, particularly, for example, in the peroxidase method of white blood cell differential counting performed on automated hematology analyzers.
Thus, there is a clear need in the art for improved rinse reagent solutions or diluents for use in methods in which blood samples are processed and analyzed using automated hematology systems. Such reagent solutions and methods using such reagent solutions are needed to thoroughly cleanse all of the reagent chambers, the channels, and other mechanical hardware of the automated analyzers (e.g., the system hydraulics, including pumps, tubing, valves, and the like) following aspiration of the samples from the chambers to remove residual debris and to alleviate the formation of buildup in the hydraulic path. Also, such reagents and methods would fulfill the need to maintain the optimum integrity of the resulting cytograms after many rounds of sample analysis. Also needed in the art are reagents that are capable of simplifying the design and operation of automated hematology systems, which are frequently quite versatile and suitable for several different types of hematology methods and procedures for performance on such systems. A further need in this art is the elimination of unecessary redundancy of reagents, particularly rinse reagent solutions, for economy and for the streamlining of blood sample analyses.
Reagent compositions have historically been developed for use in automated systems for very specific purposes. As is conventional in the art, a reagent composition formulated, tested, and used over time to achieve a particular purpose and to perform a particular function in a system, is recognized and routinely used in the art solely for that purpose and function in that system. Thus, under routine circumstances, a reagent composition, which is optimized for a particular purpose and function, is appreciated, used, and most frequently taken for granted by those in the art based on its known and intended purpose and for no other purpose. It is only rarely and unexpectedly that a reagent or material designed and used for a specific purpose in the art happens to be found useful in a completely different way and/or for a completely different and unique purpose that is unrelated to its original and intended use. Such a new use or application of a reagent is neither expected nor predicted by those having skill in the art.
An example of one reagent composition used routinely to perform a particular function on automated hematology systems is known as a red blood cell/basophil sheath ("RBC/Baso sheath"). The RBC/Baso sheath was designed for use on the abovementioned automated analyzers of the TECHNICON H.circle-solid..TM. series to surround the sample stream by a concentric layer of liquid to prevent the cells in the reaction mixture sample streams of the red blood cell and basophil channels from contacting or touching the walls of the analyzer flow cell. The sheath is thus a "passive" or noninteractive reagent, since it does not physically interact with blood cells to any significant degree; it was not designed or used to contact samples. The sheath contains a surfactant in order to prevent the formation of bubbles which interfere with the automated methods by causing the sample stream to wander out of alignment, thereby producing distorted optical registration by the detector. Other ingredients in the RBC/Baso sheath reagent are phosphate buffered saline having an osmolality of about 290 mOsmol/kg and an antioxidant to protect the surfactant from autooxidation. The osmolality of the sheath reagent was designed to be isotonic to red blood cells so that their mean cell volumes would not change if there was inadvertent contact between blood cells in the sample stream and the sheath surrounding the sample stream. A preferred surfactant, Pluronic.RTM. P105, is present in the sheath reagent at a concentration that is nonlytic to red blood cells, in case there was unexpected contact between cells in the sample stream and in the surrounding sheath stream.
The RBC/Baso Sheath conventionally operates in a closed system as follows: the Sheath is introduced into the RBC/Baso flow cell by negative pressure from a syringe pulling the sheath out through the top of the flow cell, at the same time that a larger positive pressure diaphragm pump delivers the sheath through a concentric flow module at the bottom of the flow cell. The sample stream enters the concentric flow module at a different point. The velocities of the optically transparent sheath fluid and the sample stream (which have the same refractive indexes) are controlled so that laminar flow (i.e., non-turbulent) conditions exist. The sample and the sheath streams flow independently through the flow cell. It is the hydraulic pressure of the sheath stream that constricts the sample stream to its appropriate diameter. Thus the sheath performs its function of protecting the sample stream from touching the parts of the automated system hardware.
However, prior to the present invention, neither the RBC/Baso sheath nor other reagents employed to perform their particular functions in automated hematology analyzers have been recognized or used for the purpose of a rinse solution having universal applicability to all types of blood cell analysis. Thus, there is still a need extant in the art for providing a universally applicable reagent that can be used as a rinse reagent to keep systems free of buildup over extended periods of continous and varied operations. The use of such a widely-acceptable reagent promises to simplify and streamline the designs of current systems, to alleviate background noise caused by unwanted cell debris generated during the performance of hematology methods, and to generally improve the operativity of automated systems used in the field of blood sample analysis.